Relationship Of The CHFR Methylation And Microtube Inhibitors Sensitivity In Esophageal Cancer

Background and objects:CHFR(checkpoint with FHA and ring finger) is a new cell cycle checkpoint gene,which is inactivated in human cancers. During stress mitosis,it has the function of delaying chromatin condensation and progression to the mitotic phase.Some research results show that, in many tumors,CHFR expression silencing because of the CpG island methylation. In tumor cells CHFR silencd by methylation are often associated with sensitivity to microtubule inhibitors. In this study, methylation specific PCR (MSP) was detected the methylation of the promoter region of CHFR gene and the epigenetic control mechanism about CHFR gene in normal esophageal mucosa, esophageal dysplasia, esophageal cancer and esophageal cancer cell lines. Use RT-PCR analysised the expression of CHFR mRNA in esophageal cancer cell lines, before and after use 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor),to find the relationship between CHFR expression and CHFR promoter region hypermethylation.With various chemotherapeutic agents treatment of esophageal cancer cell lines, by detecting cell activity and cell cycle,to analysised chemosensitivity in esophageal cancer cell lines.Methods:part I. RT-PCR, detection the expression of CHFR gene before and after 5-aza-dc treatment of esophageal cancer cell lines for further clarify whether the expression of CHFR gene by epigenetic regulation. Using MSP detected the methylation of CHFR gene in normal esophageal mucosa, esophageal dysplasia, esophageal cancer and esophageal cancer cell lines,.Part II.With Tax, Doc, VP16, CDDP,5-aza-dc treated esophageal cancer cell lines and detected the cell activity by CCK-8 kit in methylated cell lines (KYSE70, KYSE150) and unmethylated cell lines (KYSE140, KYSE450), according to cell growth inhibition rate to determine its sensitivity to chemotherapeutic drugs. Cell cycle detected by flow cytometry in methylated cell line (KYSE70, KYSE150) and unmethylated cell line (KYSE450) after 5-aza-dc, Tax, Doc, VP16 or CDDP treated, further study the mechanisms of chemotherapy sensitivity.Result:The expression of CHFR mRNA was detected by RT-PCR.In cell lines KYSE30, KYSE140, KYSE180, KYSE450, KYSE520, TE7 and SEG1, mRNA of CHFR were expressed,but in cell lines KYSE70, KYSE150 and KYSE510 were loss of expression. In order to verify the mRNA expression of CHFR is associated with methylation, we applied methylation inhibitor 5-aza-dc treatment of esophageal cancer cell lines for 96h, the expression found previously missing in KYSE70, KYSE150 and KYSE510 was recovery, while the previous expression of other cell lines were unchanged. These results suggest that 5-aza-dc can reverse the expression of CHFR gene silencing and restore mRNA of CHFR gene.MSP results showed that, hypermethylation of CHFR promoter region was not found in 6 cases of normal esophageal mucosa, but was found frequently methylated in primary esophageal cancer (49/109,50%).By contrast, CHFR promoter region methylation is rare event in esophageal dysplasia (2/50, 4%).Significantly different from the three (p<0.05).with clinical and pathological data of patients,no correlation was found between hypermethylation and gender, age and stage(P> 0.05).Cell activity:the Inhibitory rate of cell growth of taxanes or docetaxel treatment are higher in methylated esophageal cancer cell lines (KYSE70, KYSE150) than in unmethylated esophageal cancer cell lines (KYSE140 and KYSE450) (p<0.05). The Inhibitory rate of cell growth of methylated cell lines (KYSE70 and KYSE150) is lower in 5-aza-dc+taxanes or 5-aza-dc+docetaxel treatment than in taxanes or docetaxel treatment group (p<0.05) for 24 hrs and 48 hrs, but no difference was found at 72 hrs. For unmethylated esophageal cancer cell lines (KYS140 and KYSE450), the Inhibitory rate of cell growth is higher in 5-aza-dc+taxanes or 5-aza-dc+docetaxel treatment than in taxanes and docetaxel treatment group (p<0.05). After 5-aza-dc treatment the Inhibitory rate of cell growth are higher than before 5-aza-dc treatment in CDDP or VP16 treatment group for all cell lines(p<0.05).Flow cytometry cell cycle:0.00% of KYSE70 and KYSE150 cells were in G2-M phase by only Tax or Doc treatment. But after Tax+5-aza-dc or Doc +5-aza-dc treatment, G2-M phase cells were increased aparrently in methylated cell lines(KYSE70 and KYSE150) (p<0.001). For CHFR unmethylated cell line (KYSE450), G2-M phase cells were increased apparently after Tax or Doc treatment. But after Tax+5-aza-dc or Doc+5-aza-dc treatment, there are no significant changes on G2-M phase cells for CHFR unmethylated cell line KYSE450)(p>0.005).Conclusion:CHFR gene in the pathogenesis of esophageal cancer has an important role,its promoter region CpG island methylation leads to inactivation of CHFR inhibits its expression. Application of 5-aza-dc treatment restored CHFR expression of esophageal cancer provides a new way of another therapy. According to the relationship between methylation of CHFR with chemotherapy drugs in esophageal cancer cell lines, we hypothesized that CHFR methylation is highly relevant to chemosensitivity of microtubule inhibitors in esophageal cancer cells, assessment of CHFR methylation status may be a clinically useful marker to predict the response of esophageal cancers to microtubule inhibitors.